Method of removing shiga-like toxins from biological samples

ABSTRACT

Diagnostic and therapeutic compositions which comprise the αGal(1-4)βGal subunit are described. These compositions permit the rapid diagnosis and treatment of enteric infections caused by E. coli that produce shiga-like toxins (SLT).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 07/778,732 filed Oct. 18, 1991, now abandoned, which application is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The invention relates to diagnosis and treatment of diarrhea caused by bacterial infection. More specifically, the invention concerns detection and neutralization of shiga-like toxins (SLT) associated with enteric bacterial infection.

BACKGROUND ART

Diarrhea caused by strains of pathogenic E. coli has been found to be associated with the production of a variety of enterotoxins. Some pathogenic E. coli produce enterotoxins that are closely related to the shiga toxin associated with Shigella-caused dysentery. The first member of the family of shiga-like toxins (SLT) to be isolated was cytotoxic for African Green Monkey (Vero) cells and was originally called verotoxin. Since its structural similarity to shiga toxin has been established by sequencing of the relevant genes, this toxin is now more commonly called shiga-like toxin I (SLTI). See, for example, Calderwood, S. B., et al., Proc Natl Acad Sci USA (1987) 84:4364-4368; Jackson, M. P., et al., Microb Pathog (1987) 2:147-153; Strockbine, N. A., et al., J Bacteriol (1988) 170:1116-1122.

Additional members of the SLT family have subsequently been isolated that can be distinguished serologically, on the basis of gene sequence or host specificity (Gannon, V. P. J., et al., J Gen Microbiol (1990) 136:1125-1135; Weinstein, D. L., et al., J Bacteriol (1988) 170:4223-4230; Ito, H., et al., Microb Pathog (1990) 8:47-60; Head, S. C., et al., FEMS Microbiol Lett (1988) 51:211-216; Schmitt, C. K., et al., Infect Immun (1991) 59:1065-1073; Scotland, S. M., et al. Lancet (1985) ii:885-886; Oku, Y., et al., Microb Pathog (1989) 6:113-122. Various types of SLTII have been described and have been assigned various designations depending on the strain of E. coli from which they are isolated and the hosts they inhabit. Thus, variants have been designated SLTII; vtx2ha; SLTIIvh; vtx2hb; SLTIIc; SLTIIvp and so forth.

All of the SLTt's are multimeric proteins composed of an enzymatic (A) subunit and multiple (B) subunits responsible for toxin binding to receptors on host tissues. The binding B oligomers of SLTI, SLTII and SLTIIvh recognize host cell globoseries glycolipid receptors containing at a minimum the disaccharide subunit αGal(1-4)βGal at the non-reducing terminus; SLTIIvp has been shown to bind to the receptors containing this subunit but not necessarily at the non-reducing end (Samuel, J. E., et al., Infect Immun (1990) 58:611-618; Boyd, B., et al., Nepbron (1989) 51:207-210; DeGrandis, S., et al., J Biol Chem (1989) 264: 12520-12525; Waddell, T., et al., Biochem Biophys Res Comm (1988) 152:674-679; Lingwood, C. A., et al., J Biol Chem (1987) 262:8834-8839; Waddell, T., et al., Proc Natl Acad Sci USA (1990) 87:7898-7901; Cohen, A., et al., J Biol Chem (1987) 262:17088-17091; Jacewicz, M., et al., J Exp Med (1986) 163:1391-1404; Lindberg, A. A., et al., J Biol Chem (1987) 262:1779-1785).

SLT activity has been detected in stool samples of symptomatic patients (Karmali, M. A., et al., J Clin Microbio1 (1988) 22:614-619; Maniar, A. C., et al., J Clin Microbiol (1990) 28:134-135). However, there is difficulty in detecting the presence of SLTs clinically since these are very potent toxins present in low concentrations. In order to assure detection, the SLT present in the sample must be concentrated to enhance the reliability of the assay. Present diagnostic procedures are technically demanding, time consuming and of limited practical use in the clinical setting. Thus there is a clear need for improved diagnostic clinically practical and rapid procedures.

Also, antibiotics are not recommended for treatment of enterohemorrhagic E. coli infection (Robson, W. L. M., et al., J Pediatr (1990) 117:675-676) and the use of antimotility drugs also appears to be counterproductive (Cimolai, N., et al., J Pediatr (1990) 117:676. There is, therefore, also a clear need for new and effective therapeutic agents.

It has now been found that artificial substrates containing the αGal(1-4)βGal(P₁ disaccharide) subunit and more preferably the αGal(1-4)βGal(1-4)βGlcNAc (P₁ trisaccharide) or αGal(1-4)βGal(1-4)βGlc (P_(k) trisaccharide) subunit are effective in detecting and neutralizing members of the SLT family under conditions necessary to effect recovery of the patient and as such represent novel therapeutic and diagnostic tools in the treatment of E. coli -mediated dysentery.

DISCLOSURE OF THE INVENTION

The invention provides compositions and methods that permit practical and effective diagnosis of E. coli-caused enterotoxic bacterial infections that present clinically as severe diarrhea, hemorrhagic colitis, hemolytic uremic syndrome or thrombotic thrombocytopenic purpura. The invention also provides compositions useful in the therapy of these and related conditions.

Thus, in one aspect, the invention is directed to a method to simply and rapidly bind to a support shiga-like toxins (SLT) from a biological sample at physiological conditions for diagnostic use, which method comprises contacting said sample with an effective amount of an affinity support wherein the affinity ligand comprises an αGal(1-4)βGal subunit under conditions wherein said SLT is adsorbed to the affinity support; and detecting any SLT bound to the support.

In another aspect, the invention is directed to methods to detect the presence of SLT in a biological sample which method comprises contacting said sample with a composition comprising the αGal(1-4)βGal subunit under conditions wherein said subunit is complexed to any SLT present in the sample and detecting the presence of any complex formed.

In a third aspect, the invention is directed to a method to treat enteric infections caused by microorganisms that produce one or more SLTs, which method comprises administering to a subject in need of such treatment an effective amount of a composition comprising the αGal(1-4)βGal subunit.

In still other aspects, the invention is directed to pharmaceutical compositions which comprise the αGal(1-4)βGal subunit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A and B demonstrate the toxicity of bacterial extracts obtained using polymixin-B with respect to their ability to kill Vero cells in the presence and absence of various SYNSORBs.

FIGS. 2 A and B demonstrate the toxicity of bacterial extracts obtained using lysozyme with respect to their ability to kill Vero cells in the presence and absence of various SYNSORBs.

FIG. 3 A demonstrates that as little as 5 mg of P_(k) trisaccharide SYNSORB removes >90% of SLT toxins from bacterial extracts.

FIG. 3B demonstrates that the binding of the SLT toxins occurred within 5 minutes of mixing extracts with the P_(k) SYNSORB.

FIG. 4 demonstrates the difficulty in eluting the bound I¹²⁵ labelled SLTI from various SYNSORBs utilizing a variety of eluants.

FIG. 5 demonstrates that >90% SLTI, SLTII/IIc and SLTII activity was neutralized by co-incubation of Vero cells and SLT extracts for three days, with as little as 10 mg of P_(k) trisaccharide SYNSORB.

The subunit is bound preferably through the --(CH₂)₈ C(O)-- linking arm as that described by Lemieux, R. U., et al., J Am Chem Soc. (1975) 97:4076-4083; to a solid, inert support that can be easily eliminated from the gastrointestinal system. Inert silica matrix embodiments which are commercially available as "SYNSORB™" are preferred.

MODES OF CARRYING OUT THE INVENTION

The compositions useful in the conduct of the methods of the invention include a αGal(1-4)βGal disaccharide subunit, preferably the αGal(1-4)βGal (1-4)βGlcNAc trisaccharide subunit or αGal(1-4)βGal (1-4)βGlc trisaccharide subunit, preferably at the non-reducing terminus of an oligosaccharide. The di- and trisaccharides may be provided as a portion of a larger oligosaccharide coupled to a solid support or coupled directly, preferably through a linking arm such as that described by Lemieux, R. U., et al., J Am Chem Soc (1975) 97:4076-4083. The di- and trisaccharide subunits may also be coupled directly to pharmaceutically acceptable carriers or constitute a portion of an oligosaccharide coupled to such carriers. Depending on the application for which the compositions of the invention are suggested, the composition is designed to accommodate the di- or trisaccharide subunits so as advantageously to be employed in these applications.

As used herein, "shiga-like toxin" or "SLT" refers to group of toxins produced by enterohemorrhagic E. coli that resemble the Shigella-produced shiga toxins as is commonly understood in the art. These toxins comprise an enzymatically active A subunit and a multimeric receptor binding B subunit. Such SLTs include SLTI and the various grouped toxins designated in the art SLTII.

Rapid, tight binding of SET's to P₁ disaccharide, P₁ trisaccharide or P_(k) trisaccharide is demonstrated by the verocytoxicity neutralization and I¹²⁵ binding assays contained herein. SYNSORB™ bearing haptens, e.g., the G_(M1) ganglioside Neu5-Ac(2-3)βGal(1-4)βGlc and heat labile toxin from enterotoxigenic E. coli. would be expected to behave similarly. A single SYNSORB™ bearing several haptens with specificity for the different binding subunits of several different gastrointestinal infections should now be possible. Such universal SYNSORB™s would provide rapid, simple simultaneous diagnosis of a variety of gastrointestinal disorders.

The SYNSORB™s employed were obtained from Chembiomed (Edmonton, Canada). In each case the 8-methoxycarbonyloctyl glycoside of the respective hapten is activated and ligated to a silylaminated solid support followed by acetylation of the remaining amine groups on the solid support. These formulations are sold commercially as "SYNSORB™"s:

"P₁ -di," which contains 0.60 μmole/g αGal(1-4)βGal disaccharide;

"P₁ -tri," which contains 0.91 μmole/g eGal(1-4)βGal(1-4)βGlcNAc trisaccharide;

"P_(k) -tri," which contains 0.74 μmole/g αGal(1-4βGal(1-4)βGlc trisaccharide;

"Linear B like tri," which contains 0.47 μmole/g αGal(1-3)βGal(1-4)βGlcNAc trisaccharide;

"Linear B like di," which contains 0.66 μmole/g αGal(1-3)βGal disaccharide;

"Glucose mono," which contains 1.0 μmol B-glucose; 8-methoxycarbonyoctanol activated and ligated to the silylated solid support.

A major aspect of the invention is the rapid efficient binding of physiological concentrations of any SLT present in biological samples, thus permitting assay of quantities of these toxins. Typically, in view of the conditions with which these toxins are associated, the biological sample will be a stool sample. The sample is extracted and prepared using standard extraction techniques and the extract is contacted with a solid support derivatized to an affinity ligand, wherein the affinity ligand comprise the αGal(1-4)βGal disaccharide subunit, preferably the αGal(1-4)βGal(1-4)βGlcNAc trisaccharide subunit or αGal(1-4)βGal(1-4)βGlc trisaccharide subunit. Said contact may be in a batch treatment of the sample extract with the solid support, or the solid support may be supplied as a chromatography column and the sample extract applied to the column under conditions wherein any SLT present in a sample is absorbed.

SLT may be measured directly on the surface of the SYNSORB™ using any suitable detection system. In one approach, monoclonal or polyclonal antibodies specific for SLT can be utilized to quantify the amount of SLT bound directly to SYNSORB™, labeled, for example, by radioactive, biotinylated, or fluorescent moieties. A wide variety of protocols for detection of formation of specific binding complexes analogous to standard immunoassay techniques is well known in the art.

Compositions containing the αGal(1-4)βGal disaccharide subunit, preferably the αGal(1-4)βGal (1-4)βGlcNAc trisaccharide subunit or αGal(1-4)βGal (1-4)βGlc trisaccharide subunit, that can also be used as therapeutic agents may be supplied wherein the disaccharide subunit or trisaccharide subunits or an oligomer saccharide containing it is coupled to a nontoxic carrier, such as a liposome, biocompatible polymer, or carrier analogous to the above-referenced SYNSORB™s.

Alternatively, the disaccharide or a larger moiety containing it as a subunit may be formulated in standard pharmaceutical compositions for administration to the patient. Typically, the patient will be afflicted with a diarrhetic condition, and the target SLT will be present in the intestinal tract. Thus, a suitable mode of administration is through oral administration or other means of direct application to the gastrointestinal tract. The correct dosage range will depend on the severity of the infection, the mode of administration, the mode of formulation, and the judgment of the attending practitioner.

Activity of the SLTs can be assayed by taking advantage of the toxicity of these compounds to Vero cells. Vero cells (ATCC CCL81) can be obtained from the American-Type Culture Collection, Rockville, Md. These are maintained at 37° C./5% CO₂ in minimal essential medium with Earl's salts (MEM, Gibco BRL, Gaithersburg, Md.) containing 3% fetal bovine serum (FBS). Confluent Vero cell monolayers are disrupted using 0.25% tissue-culture grade trypsin and approximately 10⁵ cells in 200 μl FBS-supplemented MEM are added to each well of a 96-well microtiter plate. The plates are incubated overnight at 37° C./5% CO₂.

The samples to be tested and suitable controls are added to the various wells and the plates are incubated for 2-3 days at 37° C./5% CO₂. Cytotoxic effects are readily visible in successful candidate wells as compared to control wells. The results can be quantitated by aspirating the remaining liquid from each of the wells and fixing the Vero cells which remain viable with 95% methanol and staining with Geimsa stain. The results are recorded using a microtiter plate reader set at a wavelength of 620 nm, as described by Samuel, J. E., Infect Immun (1990) 58:611-618 (supra). The absorbance data are plotted versus the logarithm of the dilution of the candidate test solution. The dilution of samples resulting in 50% destruction (CD₅₀) of the monolayers is determined by extrapolation from the resulting Vero cell killing curves.

The following examples are intended to illustrate but not to limit the invention.

EXAMPLE 1 SYNSORB--Verocytotoxicity Neutralization Assays

E. coli strains 0157:H-(E32511), which produces SLTII/SLTIIc and O26:H11 (H19) which produces SLTI only, or strain C600(933W), which produces SLTII only, were grown overnight at 37° C. on tryptic soy broth (Difco, Detroit, Mich.) agar plates. Polymycin and lysozyme extracts were prepared as described previously [Karmali, M. A., et al., J Clin Microbiol (1985) 22:614-619 and Head, S., et al., Infect Immunol (1990) 58:1532-1537)].

The first neutralization assay was designed to test the ability of SYNSORBs to absorb SLT activity from the E. coli extracts. The assay involved incubating 1 mL of the E. coli extracts for 30 min. at room temperature in 1.5 mL microcentrifuge tubes (Fisher) with 2 to 50 mg SYNSORB on an end-over-end rotator. The tubes were then removed from the apparatus and after the SYNSORB had settled to the bottom (a few seconds), serial five-fold dilutions of the absorbed extracts were prepared in unsupplemented MEM. Twenty (20) μL of each dilution was added to the appropriate wells in 96 well microtiter plates containing Vero cells. Bacterial extracts to which no SYNSORB was added served as controls. Once cytotoxic effects became apparent (2 to 3 days in the incubator) the growth medium was aspirated from each of the wells and Vero cells which remained viable were fixed with 95% methanol and stained with Giemsa stain (Fisher). The results were then recorded using a microtiter plate reader set at a wavelength of 620 nm as described previously [Samuel et al., Infect Immun. 58:611-618 (1990)]. The absorbance data were then plotted versus the logarithm of the extract dilution. The dilution of the extracts resulting in 50% destruction (CD₅₀) of the monolayers was determined by extrapolation from the resulting Vero cell killing curves. Individual experiments were always performed in duplicate and, unless otherwise indicated, repeated at least two times. The percentage of neutralization was computed from the equation: 100-(100[CD₅₀ oligosaccharide SYNSORB-treated extracted+CD₅₀ acetylated silyl-aminated (ASA) SYNSORB-treated extract]). The non-parametric Mann-Whitney test using the two-tailed statistic T was employed to compute the significance level of difference between groups of independent observations [Altman, D. G., Practical statistics for medical research, 1st ed. New York, Chapman and Hall: 179-228 (1991)].

The second neutralization assay (co-incubation assay) was designed to test the ability of P_(k) trisaccharide SYNSORB to protect Vero cells from SLT activity over 3 days at 37° C. This assay involved incubating 180 μL of serial five-fold dilutions of polymyxin extracts in ethylene oxide-sterilized 1.5 mL microcentrifuge tubes each containing 2, 5 or 10 mg of P_(k) trisaccharide SYNSORB. After 1 h incubation with SYNSORB, the entire contents of each microcentrifuge tube were added to Vero cells monolayers in microtiter plates prepared as described above. The microtiter plates prepared as described above. The microtiter plates were then incubated at 37° C. for 3 days and the results of the experiment were recorded as described above (FIGS. 1 and 2).

The foregoing determination was repeated using varying amounts of Pk-tri and various times of incubation, with the results shown in FIGS. 3A and 3B. As shown in FIG. 3A, as little as 5 mg SYNSORB was capable of neutralizing the activity of the extracts of both E32511 and H19 strains; similarly, as shown in FIG. 3B, only about 5 min incubation was required to achieve this result in either extract.

EXAMPLE 2 Iodinated SLT I Binding Assay

Purified SLTI was iodinated in 12×75 mm acid-washed glass culture tubes coated with 40 μg of IODO GEN (1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril) (Pierce Chemical Co., Rockford, Ill.). About 6 μg of purified SLTI was incubated for 1 min with 20 MBq ¹²⁵ -I labeled sodium iodide in 100 μl PBS. The reaction mixture was passed through a glass wool-plugged Pasteur pipette into 200 μl PBS containing a solution of cysteine (1 mg/ml) in PBS as described by Armstrong, G. D., et al., Infect Immunol (1987) 55:1294-1299. After 1 min, 200 μl of PBS containing 1% BSA was added to the mixture and the iodinated SLTI was purified by passing the solution through a 1 cm×30 cm SEPHADEX-G 25 gel filtration column with 0.1% BSA in PBS. The efficiency of the iodination reaction was determined by measuring the number of counts that were incorporated into trichloroacetic acid precipitated protein. Aliquots of the iodinated SLTI were stored at -90° C.

The assays were performed in PBS containing 0.1% BSA to reduce nonspecific binding. 2 mg of the various SYNSORBs were incubated for 30 min on an end-over-end rotator with approximately 20,000 dpm of the iodinated SLTI prepared as above (specific activity, 2.2×10⁷ dpm/μg, CD₅₀ in the Verocytotoxicity assay, 0.4 pg/ml), in 0.5 ml PBS/BSA). The SYNSORBs were then washed with 3×1 ml portions of PBS BSA to remove unbound counts. The derivatized SYNSORBs were counted in an LKB Rackgamma model 1270 Gamma Counter.

The results are shown in Table 1.

                  TABLE 1                                                          ______________________________________                                         SYNSORB       % SLTI Bound                                                     ______________________________________                                         Pk-tri        93                                                               # 115         21                                                               Glc           9                                                                ASA           5                                                                ______________________________________                                    

The SLT bound to Pk-tri SYNSORB could be partially released using 0.1M acetic acid, 6M guanidine HCl, or by heating in boiling water bath for 30 min in 10% SDS. However, neither 0.5M lactose, 0.5M galactose, or 0.2M EDTA could displace the bound SLTI (FIG. 4).

Subsequent experiments showed that 2 mg of Pk-tri neutralized approximately 90% of the activity in E. coli H19 (SLTI) but about 10 mg Pk-tri SYNSORB was required to neutralize the activity of the E. coli 32511 (SLTII/SLTIIc) or E. coli C600/933W (SLTII) to a similar extent (FIG. 5). 

We claim:
 1. A method to bind and remove shiga-like toxins (SLT) from a liquid comprising a biological sample, which method comprises:mixing said liquid with a solid, inert affinity support having an affinity ligand covalently attached to the support through a --(CH₂)₈ C(O)-- linking arm wherein said ligand is an oligosaccharide containing the disaccharide subunit αGal(1-4)βGal at the non-reducing terminus which binds the SLT, wherein said mixing is conducted under conditions wherein said SLT binds to the affinity support; and separating the support containing the subunit to which said SLT is bound from the liquid.
 2. A method to bind and remove shiga-like toxins (SLT) from a liquid comprising a biological sample, which method comprises:mixing said liquid with a solid silica support containing affinity ligand covalently attached to the support through a --(CH₂)₈ C(O)-- linking arm wherein said ligand is an oligosaccharide selected from the group consisting of αGal(1-4)βGal--, αGal(1-4)βGal(1-4)βGlcNAc-- and αGal(1-4)βGal(1-4)βGlc--, wherein said mixing is conducted under conditions so that said SLT binds to said oligosaccharide; and separating the support containing the oligosaccharide to which said SLT is bound from the liquid. 